Control of acidic gut syndrome

ABSTRACT

According to the present invention there is provided a method for the treatment or prophylaxis of acidic gut syndrome resulting from the accumulation of acid and production of endotoxin in the gastrointestinal tract of a human or an animal, said accumulation resulting from the fermentation of carbohydrate in the gastrointestinal tract of said human or animal, wherein said method comprises administering to said human or animal an effective amount of an active agent capable of preventing or controlling acid and endotoxin accumulation in the gastrointestinal tract.

TECHNICAL FIELD

This invention relates to the treatment or prophylaxis of conditionsresulting from acidic gut syndrome in humans or an animals, whereinacidic gut syndrome results from the fermentation of carbohydrate in thegastrointestinal tract of the human or animal. The problems associatedwith acidic gut syndrome, and overcome with treatment, include:predisposition to ulceration of the gastrointestinal tract; ulcerationof the stomach; immune conditions associated with localised inflammationof the gut including irritable bowel disorder, crohn's disease,appendicitis, colitis and reduced feed intake, responsible for cachexiaand low efficiency in production feeding systems; dermatitis; arthritis;rheumatoid arthritis; osteoarthritis; respiratory tract disorders,including asthma and predisposition to bleeding in lungs followingstrenuous exercise; predisposition to microbial and helminth infectionsof the gut, and infection of the mammary gland, including mastitis;immune disorders causing predisposition to infection by bacteria, fungior protozoa; cystic fibrosis and certain cancers; effects on thepancreas, kidneys, thyroid and other organs and conditions of theendocrine system, including diabetes; homeostasis disorders, includingblood pH, mineral and electrolyte imbalances, such as osteoporosis andhypertension; immune disorders, including multiple sclerosis,amyotrophic lateral sclerosis, chronic fatigue syndrome, myastheniagravis, Alzheimer's disease, impaired reproductive performance; dentalcaries; viral infections, including herpes; exacerbation of heat stress;and impaired hair and wool growth.

BACKGROUND ART

Carbohydrates are digested through enzymic breakdown and absorption ofsimple sugars or through microbial fermentation and absorption of shortchain volatile fatty acids. A condition known as lactic acidosis(D-lactic acidosis, fermentative acidosis or carbohydrate overload) iswidely recognised in ruminants and horses. This condition is responsiblefor deaths in ruminant livestock feeding, and in horses is associatedwith the development of laminitis (Garner et al. 1987, Rowe et al. 1994)and abnormal behaviour (Johnson et al. 1998). Lactic acidosis can alsolead to diarrhoea, infections in the hind gut and skin disorders. Thepathogenesis of lactic acidosis is described as a good example ofmetabolic acidosis in which considerable amounts of lactic acid areabsorbed through the wall of the gut (Blood et al. 1983). D-lactic acidis more slowly metabolised than L-lactic acid and therefore accumulatesin the tissues where it causes severe D-lactic acidosis. These authorsalso suggest that endotoxins from gram-negative bacteria in the gut mayplay a role in the pathogenesis of lactic acidosis (Blood et al. 1983),as it is possible that these endotoxins may be absorbed as a result ofsevere structural damage to the gut epithelium which occurs during thiscondition (Krueger et al. 1986).

The present invention describes a new condition, described as acidic gutsyndrome, which differs significantly from lactic acidosis in that itdoes not involve metabolic acidosis, resulting from acid absorption fromthe gastrointestinal tract, as a factor in its pathogenesis. Acidic gutsyndrome depends entirely on acidity within the gut and the adversetoxic effects mediated through the direct effect of acid on the gut walland through effects of acidity on microbes within the gut. Acidic gutsyndrome has a range of secondary consequences which develop from theprimary effects of acidity within the gut. These secondary effects occurboth locally and systemically. Locally, the effects are through theaction of acidity and bacterial endotoxins (produced for example,through the death of gram negative bacteria under acidic conditions) onthe gut wall itself. Systemically the effects are considered to bemediated via the immune system and a range of cytokine and relatedfactors. Furthermore, it is thought that the gut wall and gut associatedlymphoid tissue play a role in acidic gut syndrome through mediating theeffects of toxins or other factors, and releasing systemically activehormones and/or agents of the immune system.

There are a number of immune system diseases, of a chronic and/or acutenature, which are of unknown aetiology. The common factor appears to bethe involvement of various cytokines and other mediators of the immunesystem. Because the aetiology of these immune-related conditions has notbeen understood they have generally been considered to be non-specificimmune diseases. Examples of immune diseases considered in this categoryare forms of arthritis, including rheumatoid arthritis, forms ofrespiratory disease and susceptibility to respiratory problems such asasthma, reduced enzyme production by the pancreas leading to some formsof diabetes, damage to kidneys resulting in mineral imbalances andhypertension, effects on the brain which can lead to secondary hormoneimbalances with respect to control of temperature regulation,reproductive functions and other key aspects of metabolic control. Theeffects mediated via the immune system (cytokine and possibly otheractivities) can also cause inflammation and damage membranes in organsand tissues remote from the sites of bacterial activity within the gut.Organs and tissues affected in this way can include the lung (whichsubsequently increases the risk of respiratory tract infection), thestomach (which increases the risk of ulcer development), the kidneys(affecting mineral retention and hypertension). There can also be localareas of non-specific inflammation such as can occur in the gut oraround the teeth. These conditions are considered to result from a minorbuild up of acidity within the gut and the release of endotoxinsassociated with the death of gram negative bacteria as acidity increasesand gram positive bacteria predominate.

The slight increases in levels of both acidity and endotoxin within thegut associated with acidic gut syndrome, effect the host via a subtleincrease in the immune challenge. The barrier between the host and thegastrointestinal microflora is extremely important in preventinginfection and/or toxaemia. The present invention describes how theimmune challenge to the host from the gut is not constant and can beincreased in response to levels of acid in the gut which have until nowbeen considered within normal limits and of no biological consequence.Increased acidity and changes in gut bacteria, particularly in relationto endotoxin production, produces a challenge which may not necessarilylead to an immediately detectable disease condition or a dramaticimmunological response in the animal. It does however pose a sufficientchallenge to the immune system of the animal or human to causemeasurable responses (for example raised levels of tumour necrosisfactor (TNF), T-cells, monocytes, interferon and cytokines, includinginterleukin-1, -6 and -8). Because these changes to the immune systemare small and in themselves do not cause symptoms or signs of diseasethey have not previously been linked to changes in the diet or digestiveprocess. Slightly elevated levels of cytokines and or TNF tend to betransitory and have previously been regarded as normal variation betweenindividuals or non-specific immune conditions, and for these reasonshave not been studied systematically with a view to determining theimportance of diet and microbial activity within the gut. There hastherefore been no previous suggestion that diet, fermentation within thegut, and subsequent acid accumulation and endotoxin production in thegut (acidic gut syndrome), are the primary causes of serious chronicand/or sporadic disease conditions of previously unknown origin.

There is a dense and diverse population of bacteria which inhabit thegastro-intestinal tract of man and animals. The concentration of thesebacteria occurs naturally in those parts of the tract where theconditions of pH are close to neutral (around pH 6.5 to 7.5) and wherefermentable substrate is available for fermentation. The bacteria in thegut poses a potential risk to the animal in terms of infection from thegut or through the absorption of microbial toxins.

The medical and nutritional literature contains information on variousapproaches used to manage the microbial population within the gut ofhumans. Two of these are summarised below.

1. The creation of a population of lactic acid producing bacteria in thegut through inoculation with probiotics such as cultures of Lactobacilliand other lactic acid producing bacteria, in the form of yoghurtcultures or specifically cultured bacteria preparations. The hypothesisbehind this practice is that the lactic acid production may excludeother, more pathogenic, bacteria from the gut.

2. Consumption of increased amounts of soluble fibre which consists ofindigestible (but fermentable) sources of starch (resistant starch) andoligosaccharides in order to provide substrates for fermentation in thehind gut. The aim of this practice is to increase the production ofbutyric acid, which has been shown, in vitro, to enhance the metabolismof the gut epithelium and reduce the risk of colonic cancer.

It is clear that these practices, now widely recommended by dietitiansand medical practitioners, are likely to increase the amount offermentable acid production and, specifically, the amount of lactic acidand are therefore likely to promote acidic gut syndrome.

The present invention describes how the elevated activity of the immunesystem, as a result of acidic gut syndrome, is sufficient to initiate,or predispose, the host to a range of secondary conditions includingimmune diseases, inflammation, infection and damage to membranes, bycytokine, or other immune system activity, and provides a method for thetreatment or prophylaxis of acidic gut syndrome.

DISCLOSURE OF THE INVENTION

According to a first embodiment of this invention, there is provided amethod for the treatment or prophylaxis of acidic gut syndrome resultingfrom the accumulation of acid and production of endotoxin in thegastrointestinal tract of a human or an animal, said accumulationresulting from the fermentation of carbohydrate in the gastrointestinaltract of said human or animal, wherein said method comprisesadministering to said human or animal an effective amount of an activeagent capable of preventing or controlling acid and endotoxinaccumulation in the gastrointestinal tract.

Typically, the active agent may be selected from the group consistingof: antibiotics, enzyme preparations, clay preparations, compounds whichslow the digesta flow rate and probiotic preparations.

A suitable active agent of the first embodiment of the invention mayinclude an antibiotic whose action is to control acid producinggram-positive bacteria.

Typically, the antibiotic may be selected from the group consisting of:a glycopeptide antibiotic, a glycolipid antibiotic, a staphylomycinantibiotic, a polypeptide antibiotic, a macrolide antibiotic, asulphur-containing peptide antibiotic, a lincosamide antibiotic,tiamulin, a nitrofuran antibiotic, a tetracycline antibiotic, apenicillin antibiotic, a polythiazole antibiotic, an ionophoreantibiotic, a cephalosporin antibiotic, a sulphonamide antibiotic, anaminoglycoside antibiotic, a quinalone antibiotic, streptograminantibiotic, and any other antibiotic active against gram-positivebacteria responsible for the production of acid in the gastrointestinaltract.

Even more typically, the antibiotics active against gram-positivebacteria may be selected from the group consisting of:- glycopeptideantibiotics, more typically, avoparcin, teicoplanin or vancomycin;glycolipid antibiotics, more typically flavomycin (bambermycin);staphylomycin antibiotics, more typically virginiamycin; polypeptideantibiotics, more typically bacitracin zinc, bacitracin methylenedisalicylate, virginiamycin S or polymixins (B & E); macrolideantibiotics, more typically tylosin, spiramycin, virginiamycin M,josamycin, spectinomycin or erythromycin; or sulfur-containing peptideantibiotics, more typically thiopeptone, thiopeptin, sulfomycin,thiostrepton, sporangiomycin, siomycin or taitomycin; lincosamideantibiotics, more typically lincomycin or clindamycin; or tiamulin; ornitrofuran antibiotics, more typically nitrofurantoin, nitrofurazone orfurazolidone; tetracycline antibiotics, more typically chlortetracyclineor oxytetracycline; penicillin antibiotics, more typicallypenicillinase-resistant penicillins, such as oxacillin or methicillin,penicillin V or ampicillin; polythiazole antibiotics, more typicallynosiheptide; or ionophore antibiotics, more typically lasalocid,tetronasin, naracin or salinomycin; or ardacin, novobiocin sodium,bottromycin tartrate; streptogramin antibiotics, more typically,quinupristin/dalfopristin (RP 59500; Synercid) or streptogramincombinations [quinupristin/dalfopristin (RP 59500; Synercid)],everninomycin derivatives (SCH 27899), oxazolidinones (U-100572,U-100766); fluoroquinolone antibiotics, more typically, ciprofloxacin,ofloxacin, clinafloxacin, DU 6859a, grepafloxacin, levofloxacin,sparfloxacin or trovafloxacin; beta-lactam antibiotics; nitrovin(payzone), enramycin, mupiricin, margainin antibiotics, chloramphenicolsand related compounds, including florphenicol, and any combinationthereof.

A suitable active agent of the first embodiment of the invention mayinclude an exogenous enzyme preparation designed to reduce the passageof fermentable carbohydrate to the hind gut through improving thedigestion and absorption in the intestine of starches, disaccharides,oligosaccharides, non-starch polysaccharides, protein starch complexesand any polysaccharide which is incompletely digested in the intestine,but which is readily fermentable in the hind gut.

Typically preferred enzymes for the break down of non-starchpolysaccharides and starches include the following: glyconasesincluding: amylase, maltase, invertase, α-glucosidases, emulsin, andamyloglucosidase; glucanases β-glucanase, xylanase; enzymes which breakdown galactosides of the raffinosse series and other α-galactosidesincluding α-galactosidase, enzymes which break down the proteins formingpart of the matrix surrounding starches, sugars and non-starchcarbohydrates in plant material, including: pepsin, trypsin,trypsinogen, chymotrypsin and natural and synthetic proteolytic enzymesof chemical or microbial origin, enzymes which depolymerise non-starchpolysaccharides including: arabinoxylans and β-glucans, and enzymesactive in the break down of cellulose, including: cellulase, enzymesactive in the break down of colloidal polysaccharides, pecticsubstances, which include: galactouronans, galactan and arabinans, aswell as the neutral polysaccharides such as xyloglucans andgalactomannans and other non-starch polysaccharides such as:rhamnogalactouronan with arabinose and galactose, arabinogalactan,glucan, xyloglucan, galactouronan with arabinose and uronan witharabinose. These enzymes can be used individually or in combination.

A suitable active agent of the first embodiment of the invention mayinclude a clay preparation which reduces the rate of fermentation andbinds specific ions in a way which reduces the adverse effects of rapidfermentation of starch and other soluble carbohydrates in thegastrointestinal tract.

Typically, preferred clays for reducing the rate of fermentation and theosmotic effects of rapid fermentation within the gut include: kaolinite,bentonite, montmorrilonite, illite, clinoliptolite, heulandite,palygorsite, saponite, smectite, chrysotile, lizardite, talc,pyrophyllite, vermiculite, beidellite, halloysite or zeolite types ofclay, and these can be activated by a wide range of ions includingsodium, calcium, potassium and mixtures of these and other ions. Theseclays can be used individually or in combination.

A suitable active agent of the first embodiment of the invention mayinclude a compound which slows digesta flow rate, thereby increasingintestinal digestion and absorption and reducing the amount offermentable substrate passing to the hind gut.

Generally, preferred agents to slow the flow of digeseta includebiologically active peptides (BAP) in a form which will reach theduodenum, and are active in modulating the activity of the digestivetract, gastric emptying and the rate of passage through the intestine.More typically, these biologically active peptides include opioidpeptides.

Whilst a range of proteins potentially produce opioid peptides onhydrolisation, the β-casomorphins, which can be derived from β-caseinduring β-casein digestion, are particularly active.

Even more typically, the biologically active peptides includecholecystokinin (CCK), the M1 fraction of virginiamycin and the analogueof virginiamycin fraction M1, compound L-156. These biologically activepeptides can be used individually or in combination.

It has traditionally been assumed that the nutritional benefits ofproteins are only related to the essential amino acids supplied to theanimal during digestion and absorption. However through the supply ofbiologically active peptides and the production of naturally occurringopioid peptides, the rate of digesta passage is reduced and this resultsin more efficient intestinal digestion and less fermentable substratepassing to the hind gut which can contribute to acidic gut syndrome.

Practical methods of supplying biologically active opioid peptides isthrough dietary supplementation with proteins such as casein and bloodmeal. For ruminant animals the best results are obtained throughprotection of the protein against rumen degradation by polymer coatingtechnology, slow-release capsules, or through formaldehyde treatment.

A suitable active agent of the first embodiment of the invention mayinclude a probiotic preparation which reduces lactic acid accumulationby: formation of alternative end products of fermentation; production ofvolatile fatty acids rather than lactic acid during carbohydratefermentation; through increased utilisation of lactic acid; or throughthe conversion of lactic acid to volatile fatty acids which can beabsorbed from the gut, thereby reducing acidity in the gut.

Typically, preferred probiotic preparations include bacteria whichferment starch and sugars to end products other than lactic acid, (ievolatile fatty acids).

More typically, the probiotic preparations may include bacteria selectedfrom the group consisting of: Succinomonas, Butyrivibrio, Bacteroidesand Succinivibrio. These bacteria can be used individually or incombination.

Typically, preferred probiotic preparations include bacteria capable ofutilising lactic acid, and converting lactic acid to volatile fattyacids and other end products.

More typically, the probiotic preparations may include anaerobicbacteria.

Even more typically, the probiotic preparations may include bacteriaselected from the group consisting of: Megasphera, Veillenolla,Selenomonas, Propionibacterium, Anaerovibrio and Peptococcus. Thesebacteria can be used individually or in combination.

Typically, preferred probiotic preparations include yeast and mycelialpreparations capable of utilising lactic acid, and converting lacticacid to volatile fatty acids and other end products.

More typically, the probiotic preparations may include yeast andmycelial preparations such as Yea Sacc.

Typically, at least any two of the above sample microrganisms of theprobiotic preparation may be used in combination in the probioticpreparation.

According to a further aspect of the invention, the active agent mayinclude a combination of at least two of: an antibiotic, an exogenousenzyme preparation, a clay, a compound which slows digesta flow rate, ora probiotic preparation.

Compositions for administration of the active agent in the method of theinvention may be prepared by means known in the art for the preparationof compositions (such as in the art of veterinary and pharmaceuticalcompositions) including blending, grinding, homogenising, suspending,dissolving, emulsifying, dispersing and where appropriate, mixing of theactive agent, together with selected excipients, diluents, carriers andadjuvants.

For oral administration, the pharmaceutical or veterinary compositionmay be in the form of tablets, lozenges, pills, troches, capsules,elixirs, powders, including lyophilised powders, solutions, granules,suspensions, emulsions, syrups and tinctures. Slow-release, ordelayed-release, forms may also be prepared, for example in the form ofcoated particles, multi-layer tablets or microgranules.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice, sweeteners, disintegratingagents, diluents, flavourings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthangum, bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavouring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavouring. Suitable coating agents include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols, zein, shellac or gluten. Suitable preservatives include sodiumbenzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulphite. Suitable lubricants includemagnesium stearate, stearic acid, sodium oleate, sodium chloride ortalc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further include dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginateor acetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate andthe like.

The emulsions for oral administration may further include one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as guar gum, gum acaciaor gum tragacanth.

In another form the invention resides in a method of treating animals orhumans which comprises delivering to the alimentary canal a quantity ofan active agent of the form described above.

Typically the active agent can be administered by binding it to fibrousmaterials which pass undigested into the caecum, colon or other part ofthe hind gut or it can be incorporated into specially formulated feedsand foods or administered in the form of premixes, pastes, gels, gums,pellets or cubes. In one particular form of the invention,administration of the active agent to human or animal subjects is in theform of digestible capsules which release the active material into thestomach, intestine or hindgut.

The active agents may be administered with the carbohydrate feed. Theactive agent can be mixed with the feed during preparation, added to thefeed before consumption or oral administration or sprinkled on top ofthe food before it is consumed. In one particular form of the invention,in the administration of the active agent to human subjects, the activeagent can be mixed with herbs and spices coating starch based foods suchas biscuits and snack foods. Further, the active agent can be includedin pelleted feeds for animals and/or in loose mixes.

Typically, the active agent may be administered directly into the buccalcavity.

Typically, administration of the active agent to human subjects may bein the form of toothpaste which releases the active agent into thebuccal cavity.

Typically, the active agent may be administered to the human or animalsubjects via targeted delivery to the hind gut using enteric coateddelivery systems to ensure specific activity of the active agent in theterminal ileum, colon and/or caecum.

Typically, targeted delivery to different parts of the gastrointestinalsystem is achieved by multiple coatings of the active agent withmaterials sensitive to pH and/or enzyme activity, and/or microbialfermentation and/or time-dependent solubility.

Typically, a method of treating animals in accordance with the presentinvention may include delivering to the alimentary canal a quantity ofbetween 1 and 10% of total feed material of an active agent of the formdescribed above, and 90-99% feed material wherein the feed materialincludes between 1 and 10% of fibrous material. Typically the fibrousmaterial may be lucerne chaff.

The administered dose of the antibiotic can vary and will depend onseveral factors, such as the condition, age and size of the human oranimal patient, as well as the nature of the lactic acid producinggram-positive bacteria. Dosages will typically range from between 0.01and 5 mg per kg of bodyweight. More typically dosages will range frombetween 0.02 and 2.0 mg per kg of bodyweight. More typically dosageswill range from between 0.05 and 1.0 mg per kg of bodyweight. Even moretypically dosages will range from between 0.1 and 0.5 mg per kg ofbodyweight. Yet even more typically, the antibiotic is administered tothe human or animal at a rate of 0.4 mg per kg of bodyweight.

Typically, the antibiotic is administered at a rate of between 1 and 100mg per kg of dry weight of food. More typically, the antibiotic isadministered at a rate of between 1 and 75 mg per kg of dry weight offood. Even more typically, the antibiotic is administered at a rate ofbetween 1 and 50 mg per kg of dry weight of food. Yet even moretypically, the antibiotic is administered at a rate of between 5 and 40mg per kg of dry weight of food.

As above, the administered dose of the enzyme preparation can vary andwill depend on several factors, such as the condition, age and size ofthe human or animal patient, as well as the nature of the carbohydrate.Dosages will typically range from between 0.01 and 50 g/kg food drymatter. Typically, the enzyme is administered at a rate of between 0.1and 3 g per kg of dry weight of food. More typically, the enzyme isadministered at a rate of between 1 g per kg of dry weight of food.

Similarly, the administered dose of the clay preparation can vary andwill depend on several factors, such as the condition, age and size ofthe human or animal patient, as well as the nature of the carbohydrate.Dosages will typically range from between 0.5 and 100 g/kg, food drymatter. Typically, the clay is administered at a rate of between 1 and50 g per kg of dry weight of food. More typically, the clay isadministered at a rate of between 10 and 20 g per kg of dry weight offood.

Typically, the administered dose of the probiotic preparation can varybetween 10⁶ and 10¹² bacteria per kg of body weight. More typically,dose of the probiotic preparation can vary between 10⁸ and 10¹⁰ per kgof body weight.

According to another form of the invention, the active agents can beused together.

According to another aspect of the invention, the formulation of theactive agent ensures that it is administered in a palatable form to theanimal or human and in a form which retains activity and is properlymixed in the appropriate compartment(s) of the gastrointestinal tract.

Generally, the active agent is administered regularly throughout theperiod the animal or human is subjected to a high carbohydrate diet orto sugars or other fermentable compounds which are not efficientlyabsorbed prior or reaching the large intestine, colon and caecum.

More typically, the active agent is administered 1-3 times daily. Evenmore typically, the active agent is administered once daily or can beincluded in human food and animal feeds. They can be fed as powders orsuspended in water, included in pellets as well as being fed inpremixes.

More typically the active agent is mixed with the food, or is added tofeeds which contain starch or sugars which may produce an acidic patternof fermentation in the gastrointestinal tract.

A suitable treatment may include the administration of a single dose ormultiple doses. Usually, the treatment will consist of administering onedose daily of the active agent for a period sufficient to control theaccumulation of acid by fermentation of the carbohydrate in thegastrointestinal tract. Dosing may continue while sources ofcarbohydrate known to cause problems of acidic fermentation in thegastrointestinal tract are included in the diet.

More typically the active agent may be administered in a single doseimmediately before consuming meals containing sources of carbohydratewhich are poorly digested and rapidly fermented.

More typically, the active agent is administered for one day prior toand daily during the consumption of excessive quantities of food stuffscontaining readily fermentable carbohydrates.

Typically, the active agent is administered orally.

According to a second embodiment of the invention, there is provided amethod for the treatment or prophylaxis of acidic gut syndrome resultingfrom the accumulation of acid and production of endotoxin in thegastrointestinal tract of a human or an animal, said accumulationresulting from the fermentation of carbohydrate in the gastrointestinaltract of said human or animal, which method comprises immunising saidhuman or animal against the microorganisms responsible for thefermentation of carbohydrate in the gastrointestinal tract of said humanor animal.

Typically, the human or animal is immunised against bacteria whichproduce lactic acid in the gut and therefore primary agents in thedevelopment of acidic gut syndrome.

Typically, bacteria against which the human or animal is immunisedinclude: Aerococcus, Alloiococcus, Carnobacterium, Enterococcus,Lactobacillus, Lactococcus, Leuconostoc, Pediococcu, Streptococcu andTetragenacoccus among others.

More typically, bacteria against which the human or animal is immunisedinclude: Lactobacillus spp. and Streptococcus bovis type.

Even more typically, bacteria against which the human or animal isimmunised is Streptococcus bovis (Sb-5).

For example, immunisation may be achieved by intra-muscular orsub-cutaneous injection of for example, a mixture of Lactobacillus spp.and Streptococcus bovis, or either bacteria administered individually,together with a suitable adjuvant, excipient, diluent and/or carrier.

Typically, the adjuvant may include Quil A, Dex and Alum, cytokines,among others, and be of a variety of types suitable for different hostspecies.

Typically, numerous strains of Lactobacillus spp. and Streptococcusbovis are suitable, and can be cultured using carbohydrate-rich mediafrom rumen contents, caecal digesta or faeces.

Typically, a priming dose is followed by regular boosters to maintainimmunity.

Typically, the dosage rate for immunisation is between 1×10⁹ and 1×10¹¹bacterial cells per injection.

Typically, the dosage rates are approximately equivalent to between1×10⁸ to 1×10⁹ bacterial cells per kg body weight.

More typically, the dosage rates are approximately equivalent to between1×10⁸ and 5×10⁸ bacterial cells per kg body weight.

Even more typically, the dosage rates are approximately equivalent to2.5×10⁸ bacterial cells per kg body weight.

Typically, the dosage rate for immunisation of small animals, such assheep, is between 5×10⁹ and 5×10¹⁰ bacterial cells per injection.

More typically, the dosage rate for immunisation of small animals, suchas sheep, is approximately 1×10¹⁰ bacterial cells per injection.

Typically, the dosage rate for immunisation of large animals, such ascattle and horses, is between 1×10¹⁰ and 1×10¹² bacterial cells perinjection.

More typically, the dosage rate for immunisation of large animals, suchas cattle and horses, is approximately 1×10¹¹ bacterial cells perinjection.

Typically, the injection volume for sheep is between 1 mL to 3 mL, and 2to 7 mL for cattle and horses 3 to 5 mL.

More typically, the injection volume for sheep is between 1 mL to 2 mL,and 3 to 5 mL for cattle and horses.

The methods of the first or second embodiments of the invention areeffective in the treatment or prophylaxis of the conditions associatedwith acidic gut syndrome.

The conditions associated with acidic gut syndrome, and treated by themethods of the first or second embodiments of the invention, include:predisposition to ulceration of the gastrointestinal tract; ulcerationof the stomach; immune conditions associated with localised inflammationof the gut including irritable bowel disorder, crohn's disease,appendicitis, colitis and reduced feed intake, responsible for cachexiaand low efficiency in production feeding systems.

The conditions associated with acidic gut syndrome, and treated by themethods of the first or second embodiments of the invention, evenfurther include: dermatitis; arthritis; rheumatoid arthritis;osteoarthritis; respiratory tract disorders, including asthma andpredisposition to bleeding in lungs following strenuous exercise.

The conditions associated with acidic gut syndrome, and treated by themethods of the first or second embodiments of the invention, stillfurther include: predisposition to microbial and helminth infections ofthe gut, and infection of the mammary gland, including mastitis.

The conditions associated with acidic gut syndrome, and treated by themethods of the first or second embodiments of the invention, yet stillfurther include: immune disorders causing predisposition to infection bybacteria, fungi or protozoa; cystic fibrosis and certain cancers.

The conditions associated with acidic gut syndrome, and treated by themethods of the first or second embodiments of the invention, yet stilleven further include: effects on the pancreas, kidneys, thyroid andother organs and conditions of the endocrine system, including diabetes;homeostasis disorders, including blood pH, mineral and electrolyteimbalances, such as osteoporosis and hypertension.

The conditions associated with acidic gut syndrome, and treated by themethods of the first or second embodiments of the invention, alsoinclude: immune disorders, including multiple sclerosis, amyotrophiclateral sclerosis, chronic fatigue syndrome, myasthenia gravis,Alzheimer's disease, impaired reproductive performance; dental caries;viral infections, including herpes; exacerbation of heat stress; andimpaired hair and wool growth.

According to a third embodiment of the invention, there is also provideda method of diagnosing acidic gut syndrome.

Typically, the method for diagnosing acidic gut syndrome comprisesdetermining the pH of the gastrointestinal tract of said human oranimal.

More typically, the pH of the gastrointestinal tract may be determinedthrough pH indicator solutions or devices added to the toilet bowl, orpH indicator solutions, or devices, used in conjunction with stoolcollections chambers.

Typically, the pH indicator solutions may involve pH sensitive colourreagents.

More typically, the pH indicator devices, used in conjunction with stoolcollections chambers, may involve paper strips.

Even more typically, the paper strips are litmus paper.

Yet even more typically, the litmus paper may be incorporated intotoilet paper.

Typically, the acid which accumulates in the gastrointestinal tractincludes: volatile fatty acids, and/or lactic acid.

Typically, the test for acidity may involve measurement of specificacids present in the faecal material, such as volatile fatty acids,and/or lactic acid.

Typically, acid measurement is performed using specific quantitativemeasurements, wherein such measurements may be made usingchromatographic or spectroscopic techniques to measure individual acidconcentrations.

Typically, the method for diagnosing acidic gut syndrome comprisesdetermining the concentration of cytokines in the gastrointestinaltract, faeces and/or plasma of said human or animal.

Typically, the cytokine measured is selected from the group consistingof: tumour necrosis factor (TNF), interferon and interleukin.

Generally, the level of cytokine is measured via radioimmunoassay, ELISAor other molecular biological detection techniques.

Typically, the method for diagnosing acidic gut syndrome comprisesdetermining the presence of bacterial endotoxins/lipopolysaccharides inthe gastrointestinal tract of said human or animal.

The bacterial endotoxins/lipopolysaccharides may be detected throughnucleic acid hybridisation and/or amplification.

Typically, hybridisation detection occurs via a nucleic acid probespecific for a bacterial endotoxin/lipopolysaccharide.

Typically, nucleic acid amplification may also provide a method ofdetection, and may occur via a pair of nucleic acid primers specific fora bacterial endotoxin/lipopolysaccharide.

More typically, amplification may be carried out using the ligase chainreaction (LCR).

Even more typically, amplification may be carried out using thepolymerase chain reaction (PCR).

The bacterial endotoxins/lipopolysaccharides may be detected via anantibody detection system.

Typically, the detection system may involve the use of at least onepolyclonal antibody.

More typically, the detection system may involve the use of at least onemonoclonal antibody.

Typically, bacterial endotoxin/lipopolysaccharide may be measured usingthe limulus amoebocyte lysate system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the relationship between lactic acid concentrationand rumen pH.

This shows that unless lactic acid is present the pH does not fall below5.5.

FIG. 2 refers to the absorption of lactic acid and volatile fatty acids(VFA) from the rumen and caecum. This shows a net gain in lactic acidover the same period when there is significant absorption of VFA. Thesignificance of these data is that when lactic acid accumulates in thegut it is unlikely to be absorbed in the same way as the VFA, explainingwhy very low pH levels are common in the presence of lactic acid (seeFIG. 1).

FIG. 3 refers to faecal pH measured over time in a mature male consuminga diet characterised by readily digestible carbohydrate low in resistantstarch and non-starch polysaccharides for 2 weeks and then a dietconsidered to deliver higher levels of fermentable carbohydrate to thehind gut The main changes in the diet were in relation to thecomposition of the muesli (which was altered to contain higher levels ofresistant starch and non-starch polysaccharides) eaten each morning, andthe amount of pasta and beans. Arrows indicate the consumption of largepasta meals.

FIG. 4 shows that rapid carbohydrate fermentation and acidic conditionsin the gut result in endotoxin (lipopolysaccharide) production. Acidicconditions and the presence of endotoxins within the gut challenge thehost immune system via TNF, cytokine and/or other mediators to cause arange of local and system effects.

FIG. 5 shows some of the variable effects that can result from theprimary stimulation of the immune system through bacteriallipopolysaccharides. It is important to note how the effect of LPS canbe localised or can act systemically particularly so in the case of thegastrointestinal tract which is well provided with lymph nodes (GALT)able to amplify any immune stimuli from the gut. There is a far widerrange of effects than summarised in this diagram and these includetissue remodelling (collagenase, diabetes mellitus and otherproliferative and degenerative tissue conditions), coagulation system,kidney function, cachexia, appetite and others.

FIG. 6 shows pH measured in different parts of the digestive tract ofpigs fed a diet promoting hind gut fermentation (HGF diet) with orwithout virginiamycin. Experimental details are described in Example 6.It is clear from FIG. 6 that virginiamycin has a consistent effect inreducing acid accumulation (increasing pH) in all parts of the digestivetract, and provides evidence for its use in the prevention and controlof acidic gut syndrome in pigs

FIG. 7 illustrates changes in faecal pH and TNF-α measured in sheepfollowing ingestion of a pelleted diet containing a high concentrationof barley with or without virginiamycin. Experimental details aredescribed in Example 7

FIG. 8 indicates pH measured in different parts of the digestive tractof sheep following ingestion of a pelleted diet containing a highconcentration of barley hind gut fermentation (HGF diet) with or withoutvirginiamycin, wherein experimental details are described in Example 7.It is clear from FIG. 8 that virginiamycin has a consistent effect inreducing acid accumulation (increasing pH) in all parts of the digestivetract, and provides evidence for its use in the prevention and controlof acidic gut syndrome in sheep.

FIG. 9 describes faecal pH for mice given H₂O without any additives ()or given H₂O containing 5% Dextran Sulphate in solution (∘). Mice hadfree access to commercial mouse food at all times

FIG. 10 illustrates faecal pH (▪) and total disease score (□) for micegiven H₂O containing 5% Dextran Sulphate in solution. The disease scoreis based on assessment of diarrhoea, and the appearance of mucus andblood.

BEST MODE OR MODES OF PERFORMING THE INVENTION

Under certain dietary conditions, which until now have been considerednormal, increased acid and endotoxins are produced by gut microbes whichstimulate the immune-system in ways leading to local inflammation,systemic reactions related to immune diseases and perturbation ofbasement membranes in organs such as the respiratory system and thegastric stomach.

The current practices described above, do not recognise the risksassociated with increased acid production and the effect that this islikely to have on the immune system of the host. Understanding theaetiology of the new condition, acidic gut syndrome, provides afoundation on which to develop a range of treatments based on methods tominimise gut acidity associated with fermentation and thereby reduce theadverse secondary consequences mediated via the immune system.

The present invention involves four stages or levels of new informationwhich are often inter-connected and may be considered together.

Primary Effects Within the Gut

New experimental results show that lactic acid in the gut contentsproduce acidic conditions characterised by levels of pH below 5.5. Asshown in FIG. 1, the pH of the rumen does not fall below 5.5 unlesslactic acid is present. This pattern of fermentation withingastrointestinal systems applies in other parts of the gut and in otherspecies. A reason for this is the fact that lactic acid is not absorbedfrom fermentation compartments in the digestive tract in the same way asother organic acids such as acetic, propionic and butyric (Ding et al.,1998; and see FIG. 2).

Levels of acidity characteristic of acidic gut syndrome can occur in thegut under relatively normal dietary conditions. shows changes in faecalpH over time in a healthy adult male, on a normal diet experiencing nodiscomfort and passing stools of normal consistency. These data showthat the acidity of the hind gut in humans can reach levels which arealmost certain to be associated with lactic acid and bacterialendotoxins. When dietary or digestive conditions result in rapidfermentation in the gut, both the absolute level of acidity (pH) changesbut also the stability of pH. It should be emphasised that pH is a logscale and that a change from 6.8 to 5.8 represents a 10 fold increase inacid concentration. Short term changes in pH can lead to transitoryincreases endotoxins production within the gut and irregular changes inthe level of challenge to the immune system.

Many dietary conditions considered healthy for humans, dogs and otherspecies (high levels of fermentable fibre and inoculation with livelactic acid producing bacteria) result in the accumulation of lacticacid in several sections of the intestines (as shown diagrammatically inFIG. 4), wherein the low pH and bacterial endotoxins combine tostimulate the immune system of the host, and results in the diseaseconditions described above. It is important to emphasise that theseinteractions between endotoxins and low pH in the gut, to affect the gutwall can occur when normal diets are being used and when normal feedingregimes apply. Patterns of fermentation and levels of digesta pHpreviously thought to be normal and even desirable for the health andwell being of the host are therefore predisposing factors in acidic gutsyndrome in which the acidity and production of bacterial endotoxins andother potentially toxic substances have local and systemic effects viathe gut.

Local and Systemic Effects

The effects of acidic gut syndrome are mediated primarily via TNF andrelated cytokine activity but may involve other aspects of the immunesystem and/or the endocrine system. An example of the effects ofendotoxins (lipopolysaccharides) on TNF is summarised in FIG. 5. Insmall doses, cytokine activity causes local inflammation of the: gut,and with increasing doses of endotoxin, the cytokine produce numeroussystemic effects which can influence almost all tissues, and forms thebasis of many immune diseases the aetiology of which has not previouslybeen known.

The tissues most affected by systemic cytokine activity are determinedby genetic and functional factors and can also be affected by localtissue damage or physical sensitisation. Often cytokines affect morethan one organ or tissue and it is common to find simultaneous problemsfor example in the lungs and skin (asthma and dermatitis) or skin andjoint tissue (dermatitis and rheumatoid arthritis). Physical stress orirritation on joints or on other tissues can localise the action of theimmune system and its agents. In this way injuries or other irritantscan initiate reactions which are amplified by the underlying activitiesof the immune system including sub-acute inflammation. The origin ofthis elevated background immune system activity has not been previouslyunderstood and a number of disease conditions have had no known cause.The present invention therefore explains a wide range of immune diseasesincluding chronic lung conditions, sensitisation to physical, chemicaland biological irritants which cause allergic type reactions, certainforms of diabetes, problems with cartilage and connective tissue,effects on the pancreas, kidneys, thyroid and other organs of theendocrine system. It also covers immune conditions associated withlocalised inflammation of sections of the gut.

Endotoxins and the related TNF and cytokine responses can also stimulateglycolysis which, in turn, can lead to slightly elevated bodytemperature and systemic acidosis through the accumulation of L-lacticacid as a product of glucose utilisation by muscle and other tissues.This acidosis associated with TNF (Horohov et al, 1997) is not relatedto D-lactic acidosis which results from the absorption of lactic acidfrom the gut. This systemic acidosis can contribute to disruptions ofnormal mineral metabolism as the body needs to maintain anion/cationbalances as well as constant blood pH. The elevated level of tissuemetabolism associated with glycolysis can exacerbate problems of heatstress in hot and humid climatic conditions.

Since the cytokine activity also affects the brain, and via this route,the production of hormones such as prostaglandins (which are recognisedin their stimulation of pyrogenic reactions) it is likely that theadverse effects of acidic gut syndrome extend to the impairment of thereproductive system and other functions which depend on hormonal controlvia the central nervous system. Impaired reproductive performance is animportant problem in high producing dairy herds fed large quantities ofgrain to enhance milk production during early lactation (the time whenthe next mating is scheduled). It could also be a possible factorinhibiting pregnancy in certain women. This could be exacerbated by thestress and anxiety often associated with this situation, which wouldincrease digesta flow rate and deliver high levels of fermentablecarbohydrates to the hindgut, and increase the risk of acidic gutsyndrome.

Mammary development and subsequent milk production in dairy cattle isreduced under dietary situations involving grain feeding. There is alsoa high incidence of mastitis and variable feed intake secondary problemsof lameness and respiratory disease in herds fed high levels of grain.These conditions may be initiated and/or exacerbated by the acidic gutsyndrome and alleviate d by its management and prevention.

The role of TNF-α in cachexia involves, in part, a reduction in feedintake and this response was also identified in Example 6 in pigs withacidic gut syndrome. A new method of treatment of cachexia, ofpreviously unknown primary cause, can be now be treated by managementand prevention of acidic gut syndrome. Loss in efficiency due to reducedfeed intake in the production feeding of farm livestock is a seriousproblem which can be ameliorated by management and prevention of acidicgut syndrome.

Side Effects of Stress

In addition to the range of immune diseases described above there arefurther types of conditions covered in this invention which werepreviously thought to be associated with stress. One of the effects ofpsychological or physical stress is to increase the rate of digestaflow. More rapid digesta flow results in decreased residence time in theupper digestive tract and the passage of more undigested materialpassing to the lower tract. This increases the amount of fermentablesubstrate entering the hind gut and increases the risk of acidic gutsyndrome described above. Although soft faeces and diarrhoea are wellrecognised as side effects of stress, the acidic nature of the faeces(and hind gut digesta) under these conditions have not been recognisedas adverse and potentially dangerous factors which could lead to gutacidity and endotoxin production.

The effects of stress through acidic gut syndrome may have systemiceffects on other parts of the digestive tract such as the gastricepithelium which can predispose the tissue to the development of gastriculcers. This aspect of the invention is therefore based on the fact thatthe primary problem in the gut is caused by fermentation in the hind gutand not necessarily the over-producing of acid in the gastric stomach orthe presence of Helicobacter pylori. The role of acid in the gastricstomach may act to exacerbate the primary effect of systemic effects ofacidic gut syndrome.

In addition to gastric ulcers there are other adverse side effects ofstress covered by this invention including predisposition to lunginfections as a result of primary damage to the integrity of alveolarepithelium through cytokine and immune system changes initiated byacidity in the hind gut by systemic endotoxins of hind gut origin.Secondary effects also include hair and wool growth, hypertension andother factors which are mediated via the immune or endocrine system.

Furthermore, cold weather is also known to increase the rate of digestaflow. The increased risk of acidic gut syndrome is seen throughincreased incidence of arthritis and skin problems in winter.

The Effects of Acidity in the Mouth

Effects of lactic acid accumulation and microbial endotoxin productioncan occur in the most anterior portion of the gastrointestinal tract,that is, the mouth cavity itself. The teeth and gums can contain a richmicrobial population able to produce significant amounts of lactic acidwhen sugars and starches are consumed. While it is widely recognisedthat lactic acid is produced around the teeth and gums leading to dentaldecay, it is not known that microbial endotoxins are produced at thesame time. This effectively creates a form of acidic gut syndrome aroundthe gums in which acidity and endotoxins may have similar local andsystemic effects as have been described above.

The invention will now be described in greater detail by reference tospecific Examples, which should not be construed as limiting on thescope thereof.

EXAMPLES Examples 1 to 5

In general, one can refer to the accompanying FIGS. (1-5) for Examples 1to 5 of the present invention.

FIG. 1 illustrates the relationship between lactic acid concentrationand rumen pH. This shows that unless lactic acid is present the pH doesnot fall below 5.5.

FIG. 2 refers to the absorption of lactic acid and volatile fatty acids(VFA) from the rumen and caecum. This shows a net gain in lactic acidover the same period when there is significant absorption of VFA. Thesignificance of these data is that when lactic acid accumulates in thegut it is unlikely to be absorbed in the same way as the VFA, explainingwhy very low pH levels are achieved in the presence of lactic acid (seeFIG. 1).

FIG. 3 refers to faecal pH measured over time in a mature male consuminga diet characterised by readily digestible carbohydrate low in resistantstarch and non-starch polysaccharides for 2 weeks and then a dietconsidered to deliver higher levels of fermentable carbohydrate to thehind gut. The main changes in the diet were in relation to thecomposition of the muesli eaten each morning and the amount of pasta andbeans. Arrows indicate the consumption of large pasta meals.

FIG. 4 shows that rapid carbohydrate fermentation and acidic conditionsin the gut result in endotoxin (lipopolysaccharide) production. Acidicconditions and the presence of endotoxins within the gut challenge thehost immune system via TNF, cytokine and/or other mediators to cause arange of local and system effects.

FIG. 5 shows some of the variable effects that can result from theprimary stimulation of the immune system through bacterial LPS. It isimportant to note how the effect of LPS can be localised or can actsystemically. There is a far wider range of effects than summarised inthis diagram and these include tissue remodelling (collagenase, diabetesmellitus and other proliferative and degenerative tissue conditions),coagulation system, kidney function, cachexia, appetite and others.

Example 6

Acidic Gut Syndrome in Pigs

Aim:

An experiment was conducted to investigate acidic gut syndrome in thepig and the side effects mediated via the immune system. The build-up ofacid in hind gut digesta of pigs has been reported in relation toperceived benefits of increased caecal and colonic fermentation ofstarch high in amylose and of low intestinal digestibility (Brown &McNaught, 1997). However, no previous study suggests that accumulationof acid in the hind gut of pigs during fermentation of dietarycarbohydrate may, in fact be harmful. The aim of the present study wasto investigate whether acid accumulation in the hind gut is a primaryfactor leading to a wide range of adverse effects on the health andwelfare of the pig.

Hypothesis:

This experiment was based on the hypothesis that rapid fermentation ofcarbohydrate in the hind gut can lead to accumulation of acid and low pHwhich, in turn, stimulates gut cytokine activity leading to local andsystemic stimulation of the immune system with adverse effects on allsystems of the body sensitive to immune system activity. It is possiblethat the stimulation of cytokine activity by low pH in the hind gut canoccur directly through increased concentrations of acid, and/orindirectly through increased endotoxin concentrations, and it ispossible that both low pH and endotoxins act additively orsynergistically.

Explanation of Experimental Model:

It is often assumed that the digestive enzymes of the stomach and smallintestine are able to breakdown all dietary starches and sugars toglucose and that the absorptive capacity of the small intestines issufficient to ensure that no glucose passes to the caecum and colon.These assumptions are not always correct and there is practically alwayssome carbohydrate which passes undigested to the hind gut, where it isfermented and produces organic acids. The objective for the control(basal) diet was to minimise the amount of undigested starch passing tothe hind gut. For this reason boiled rice was selected, which is one ofthe most digestible sources of starch to use as the main source ofcarbohydrate for the control diets. Rice contains practically nonon-starch polysaccharides and, if properly cooked, is almost completelydigested before reaching the hind gut. In this way, hind gutfermentation of dietary starch in the control group of pigs wasminimised, and thereby minimizing acid accumulation, maintaining highpH, and minimising the risk of acidic gut syndrome.

Oligosaccharides consist of relatively short chains (3 to 10) of simplesugars in configurations not normally subject to enzymatic breakdown inthe stomach and small intestine and they pass intact into the caecum andcolon where, because of their solubility, they are rapidly fermented.Raftilose is a fructo-oligosaccharide extracted from beet root and wasselected for inclusion in those diets designed to produce rapid hind gutfermentation and an elevated risk of acidic gut syndrome. A combinationof rice and raftilose gives a semi-purified diet with the potential tovary the extent of carbohydrate digestion between the small intestineand caecum/colon. A second source of indigestible carbohydrate wasprovided by high-amylose maize. Elevated levels of amylose in maizestarch makes it less digestible in the small intestine than normal maizestarch but provides carbohydrate which is rapidly fermented in the hindgut. High-amylose maize was included together with raftilose to increasethe amount of fermentable carbohydrate entering the caecum and colon.

Studies of rapid carbohydrate fermentation and acid build up in the hindgut of sheep and horses have shown that the concentration of volatilefatty acids (VFA) starts to increase and pH starts to drop before thereis a proliferation of gram positive bacteria (particularly Streptococcusbovis and Lactobacilli) capable of producing lactic-acid. Lactic acid isa stronger acid than the VFA and is not absorbed from the caecum asquickly as VFA, and this combination of factors leads to low pH which isthe first step in development of acidic gut syndrome. Virginiamycin wasincluded in the diet of one of the two dietary treatments containingraftilose and high-maize starch to investigate its effectiveness incontrolling lactic acid accumulation and hind gut pH as a potentialmethod for preventing or controlling acidic gut syndrome.

Experimental design and treatments

There were three dietary treatments:

1 Control Boiled rice, with protein and mineral supplement to provide awell balanced diet for young weaner pigs. 2 Hind gut Boiled rice,high-amylose maize and raftilose as the fermentable sources ofcarbohydrate and a protein and mineral (HGF) supplement to provide awell balanced diet for young weaner pigs. 3 HGF diet with This dietarytreatment was identical to the HGF diet virginiamycin described aboveexcept that Eskalin Premix (Pfizer, Australia) was added to the diet toprovide 25 mg virginiamycin/kg dietary dry matter (DM).

Study Animals and Management of Pigs

Breed and No.: 18 Large White/Landrace/Duroc/Hampshire cross

Sex and age: mixed sex weaners (4 weeks) taken from two litters

Pigs were weaned at 4 weeks of age and housed individually in pens withconcrete floors with weld mesh dividing walls in temperature controlledfacilities. The pens were cleaned twice per day and drinking water wasprovided by an automatic watering system. All animals were handled dailyto acclimatise them to experimental procedures associated with sampling.

Experimental Diets and Feeding

Piglets were fed ad libitum on the basal diet for two weeks untilcommencement of dietary treatments. The diets were formulated to ensurebalanced provisions of nutrients for growth and contained 67.5%carbohydrate (rice, cornflour and raftilose) and 32.5% protein mix. Theprotein mix contained 55% meat meal, 32% fishmeal, 6% casein and 6%vitamins, oil and synthetic amino acids. The proportion of totalcarbohydrates contributed by rice, corn flour and raftilose on each dayof the study is shown in Table 1 together with the total amount offeredper pig.

TABLE 1 Amount of feed offered each day during the experimental periodand the percentage of total carbohydrate contributed by rice, corn flourand raftilose on each day. Percentage of each carbohydrate in Feedoffered HGF diet (% of DM) (g DM/pig) Rice Corn flour Raftilose Day 0301 33 45 22 Day 1 301 33 45 22 Day 2 327 27 37 36 Day 3 384 32 0 68 Day4 439 36 0 64 Day 5 521 34 0 66 Day 6 521 34 0 66 Day 7 522 27 0 73 Day8 583 22 0 78

Measurements

1. Daily collection of faeces from the pens of each pig to assess faecalconsistency and for measurement of pH. Faecal consistency was assessedon a scale 1 to 5 with 1 being liquid diarrhoea and 5 being dry,separate, well formed faeces.

2. Collections of blood from the jugular vein (10 mL) were made on themornings of days 1, 2, 6 and 8 of the study period for measurement ofIL-1, white blood cell counts and differentiation, and acute phaseprotein.

3. Body weights were measured 2 days before the start of feedingexperimental diets, on day 3 and day 7.

4. The body temperature of each animal was measured on days 0, 2, 5 and7.

5. The amount of feed not consumed was measured each afternoon andintake calculated as a % of amount offered.

6. Animals were observed daily for any signs of abnormal behaviour orill-health.

7. On day 7, half of the pigs in each treatment group were euthanasedwith pentobarbitone. Post mortem measurements were made of size of thedigestive tract, the pH of digesta in different part of the tract andthe digestive tract and other organs were examined for any sign ofabnormal tissue appearance. On day 8 the remaining pigs were euthanasedand the same post mortem measurements made on these animals.

8 Samples of digesta were stored for subsequent analysis of VFA andlactic acid concentration.

TABLE 2 Summary of results defining the build up of acid within the gutand the consequent changes in the immune system and the consequenteffects on performance and health of the animals Hind gut fermentationHGF with Prob. Control diet (HGF) virginiamycin Value Acid accumulationCaecal pH  6.4^(a)  5.3^(b)  5.7^(ab) 0.03 Rectal pH  6.8^(a)  6.1^(b) 6.3^(b) 0.01 Faecal pH (day 2)  6.9^(a)  6.1^(b)  6.3^(b) 0.03 FaecalpH (day 5)  6.5^(a)  5.9^(b)  6.0^(b) 0.05 Average faecal pH  6.7^(a) 5.9^(b)  6.0^(b) 0.001 Faecal consistency  4.65^(a)  2.89^(b)  3.53^(c)0.001 (av d1-5) Bacterial changes Caecal Lactobacilli 1.3 × 10⁹ 6.5 ×10⁹ 1.4 × 10¹⁰ Caecal Strep. Bovis 5.0 × 10⁷ 4.1 × 10⁹ 3.2 × 10⁹ No acideffect before hind gut Ileal pH  6.7  6.7  6.3 ns Stomach pH (days 8/9) 4.0  3.9  3.6 ns Changes in immune system Neutrophils (N)  6.3  9.6 6.2 0.11 Lymphocytes (L)  14.0  11.0  11.0 0.06 N:L ratio  0.47^(a) 0.95^(b)  0.57^(a) 0.02 Acute phase protein  7.5  5.8  5.9 0.11 CD3IL-1 (day 5)  1.8  2.4  2.0 ns IL-1 (av. days 1, 2, 5  2.0  2.2  1.6 ns& 8) Physiological and production effects of acidic gut syndrome Intake(% offered)  88^(a)  59^(b)  81^(ab) 0.04 (day 4) Av intake (days 1 to8)  92^(a)  78^(b)  83^(ab) 0.07 Weight change (days 3  1.18  0.73  1.130.02 to 7) Weight of washed gut (g) Stomach 135 110 115 0.09 Caecum 24.7^(a)  39.1^(b)  31.4^(a) 0.002 Rectum  25.6  40.9  35.1 0.06

The main findings of the study are described below with reference to thedata described in Table 2.

Validity of the Carbohydrate Model used to Study Acidic Gut Syndrome.

In pigs fed the HGF diet there were clear indications of acidaccumulation in the hind gut as shown by lower pH in the faeces, rectaland caecal digesta, indicating accumulation of acid as a result ofraftilose and corn flour inclusion in the diet. The lower faecal pH wasclosely related to increases in the populations of gram positive acidproducing bacteria and the incidence of diarrhoea (lower faecalconsistency scores). The fact that there were no differences due to dietin the pH of stomach or ileal digesta is of major significance. Lack ofdifference in these parameters shows that there was no accumulation ofacid (no differential effects due to fermentation of carbohydrate) priorto fermentation of raftilose and/or starch in the caecum and colon. Theeffects of the different sources of carbohydrate can therefore beattributed to the different rate and extent of bind gut fermentation onthe HGF diet leading to an accumulation of acid and a drop in pH.

As seen in Table 1, there was an increase in the proportion of raftilosein the carbohydrate fraction of the HGF diet between the beginning andthe end of the study. This decision to increase the amount of raftilosein the diet was based on the fact that on day 1 of the study there was ahigh incidence of diarrhoea in pigs on the HGF diet, whereas on day 2,faecal consistency and pH appeared normal. In order to maintain rapidfermentation and low pH in the hind gut the concentration of raftilosewas increased. This pattern of diarrhoea followed by relatively normalfaeces was repeated on days 3 and 4 and for this reason the proportionof raftilose was again increased. No adverse effects, other thandiarrhoea and low faecal pH, were observed in the pigs fed raftilose,and the nutritional model therefore appears to be well suited to thestudy of acidic gut syndrome, where the signs of hind gut acidaccumulation (mainly diarrhoea) are often taken as ‘normal’ minorproblems for which there is no cure.

Validity of the Model using Virginiamycin to Reduce Acid Build Up.

Virginiamycin was effective in reducing the extent of acid accumulationin the hind gut and in faeces (see FIG. 6). The results in relation tofaecal consistency show how the virginiamycin significantly improvedfaecal consistency. The fact that the amount of acid in the faeces andhind gut digesta, and faecal consistency of the pigs were consistentlybetween the results for pigs on the rice-based diet and those on theHGF, diet without virginiamycin, shows a clear beneficial effect ofvirginiamycin in reducing the accumulation of acid, and establishes amethod for overcoming problems in relation to acidic gut syndrome at itssource. In the present study the effect of virginiamycin in reducingacid accumulation on the HGF diet adds to the experimental design sincethere are clearly three levels of acidic gut syndrome produced by thetreatments of: rice only, HGF with virginiamycin and HGF on its own. Thefact that virginiamycin acts to reduce lactic acid production, andthereby acid accumulation, allows regression analysis of acid levelsagainst other changes in the gut physiology without complication fromthe effects of other dietary factors. On the basis that there were twolevels of lactic acid and two levels of pH and acidic gut syndrome onexactly the same diet, allows interpretation of the results on the basisof acid build up alone without complications associated with thepossible side effects of proteins and the potential allergic reactionswhich have been attributed to gluten and other proteins considered tohave harmful effects.

Immune System Responses

There were significant changes in the immune system in response to acidbuild up in the hind gut. The most notable change in the immune systemwas the twofold increase in the neutrophil to lymphocyte ratio in pigson the HGF diet. This was a result of increased neutrophil numbers and adecrease in lymphocytes. There were increases in the levels of IL-1 inpigs fed the HGF diet. The changes in neutrophil levels were apparentfrom day 1 of the study and indicate an early effect of acidic gutsyndrome on the immune system as a mechanism for effecting systemicchanges. Many changes in the immune system are transitory and it ispossible to miss peaks of cytokine and white blood cells in response tonon-continuous stimuli. A significant change in any aspect of the immunesystem is therefore an important indication of the biological pathway bywhich hind gut acid build up can affect the systemic system.

Post Mortem Findings

Two of the pigs fed on the HGF diet had clear lesions in the lungs.While this was not statistically significant it does suggest a link withHGF diets. Further, there were two clear diet related changes noted onpost mortem. These were the size and appearance of the caecum in pigsfed the HGF diet and the incidence of parakeratosis in the oesophagealzone in the stomachs of the same animals. These changes are consideredto be most significant. The changes in the caeca (increased washedweight, wrinkling upon cooling and occasional signs of inflammation andhaemorrhaging) could be explained by the increased acidity andfermentative activity within the organ. The very high incidence ofparakeratosis in pigs fed the HGF diet is significant because it is thefirst stage in development of gastric ulcers (Kavanagh, 1994), andoccurred in the absence of any changes in the pH of the stomach relatedto pH or dietary carbohydrate. It therefore appears that thediet-related damage to the oesophageal region of the stomach was causedby the primary effect of the HGF diet through rapid fermentation and abuild up of acid in the hind gut The tissue changes in the caecum inresponse to increased fermentation and consequent acidic conditions, inaddition to signs of frank inflammation in some animals, suggests thehind gut as the tissue responsible for stimulating the immune response.The rich supply of lymph nodes in the gastrointestinal mesenteryprovides the mechanism to multiply the hind gut immune response to makeit systemically significant. Changes to membranes and epithelialsurfaces are well established as consequences of acid build up in thegut of ruminants and horses. The rumen epithelium becomes detachedduring severe fermentative acidosis in sheep and cattle, and in horsesthe hoof becomes detached from the pedal bone during development oflaminitis following fermentative acidosis in the hind gut. The discoveryin this study that hind gut acid accumulation can cause parakeratosis inthe stomach (the first stage towards ulceration) provides strongevidence that acid accumulation in the gut can bring about significantchanges in membrane/epithelial structures; and that these changes canoccur in other parts of the body distant to the site of acidaccumulation.

Stomach ulcers were traditionally considered to be caused solely byelevated concentrations of acid in the stomach, and more recentlyHelicobacter pylori was identified as having an important role in thedevelopment and maintenance of stomach ulcers. The results of the studyreported here indicates an important role of the immune system inchanging the characteristics of the gut wall which creates suitableconditions for establishment of Helicobacter pylori and development ofulcers. This finding of the role of the immune system as a primaryfactor in the development of ulcers links three known predisposingfactors for the formation of ulcers.

1. Stress is well known as a predisposing factor in ulcer formation andit is also known to increase the ratio of neutrophils to lymphocytes(Puppe et al., 1997). It is likely that these and other immune systemfactors act to bring about the first changes in the gut wall whichpredispose it to the development of ulcers.

2. Certain diets are known to cause ulcers in pigs. These diets arecharacterised by having high concentrations of finely ground cerealgrain and are pelleted (Kavanagh, 1994). These features are likely todeliver substrate for extensive hind gut fermentation and acidaccumulation is likely to stimulate immune system as described above.

3. The use of antibiotics is often associated with development ofstomach ulcers. While antibiotics themselves are unlikely to causeulcers they are always used when there is an infection which can be ofsufficient severity to cause systemic changes in cytokines and otheraspects of the immune system. These infections are likely to have thesame systemic effects on the stomach as seen to arise from acidicconditions in the hind gut with both primary effects (gut acid andinfection) stimulating cytokines. A survey of pigs at slaughter showedincreased levels of neutrophils and reduced lymphocytes in animals withinflammatory conditions such as pleuritis or pneumonia (Odink et al.,1990). These changes in the immune system were similar to those observedin the current study in response to acid accumulation in the hind gut.

FIG. 6 illustrates the change in pH of digesta through thegastrointestinal tract to the faeces. It is clear from FIG. 6 thatvirginiamycin has a consistent effect in reducing acid accumulation(increasing pH) in all parts of the digestive tract, and providesevidence for its use in the prevention and control of acidic gutsyndrome in pigs and sheep.

FIG. 6 also shows that changes in faecal pH are likely to give a veryconservative estimate of the severity of acidic gut syndrome. The pH inthe caecum and proximal colon is very much lower than in the rectum orfaeces. The difference between the faecal/rectal pH and thecaecal/proximal colon is nearly one pH unit (10 fold increase).Therefore, what might appear as a relatively minor drop in faecal pHfrom 7.0 to 6.5 is likely to represent a drop in caecal pH from around6.8 down to 5.5.

Feed Intake and Liveweight Change

There were clear links between the HGF diet, consequent build up ofacid, the incidence of diarrhoea and feed intake and liveweight gain.The more severe the acidity and diarrhoea the greater the decrease infeed intake. There were no signs that the palatability of the HGF dietwas affected by incorporation of raftilose. The higher intake by pigstreated with virginiamycin (less acid accumulation and less severediarrhoea), further indicates that factors other than palatabilityreduced feed intake. While the biological pathway linking hind gut acidaccumulation and reduced intake is not clear it is possible that changesin the immune system, and particularly IL-1 could be responsible. Thisdiscovery of reduced feed intake in response to acid build up within thehind gut offer a new explanation for low, and variable, feed intake andlow efficiency in the production feeding of farm livestock.

Behavioural Changes

There were two cases of tail biting during the study and the pigsinvolved were all in the treatment groups fed the HGF diet. The bitingoccurred through the wire mesh dividing the pens. Increases in theneutrophil:lymphocyte have been found in association with increasedagonistic behaviour in pigs and again suggest involvement of the immunesystem.

Conclusions

The study provides clear evidence that acid accumulation in the hindgut, caused by rapid fermentation of undigested dietary carbohydrate,can stimulate the immune system in similar ways as observed duringinflammatory infections and stress. The only overt signs of acidicconditions in the hind gut were diarrhoea and intermittent diarrhoea isnot considered as an animal health problem. The links identified in thisstudy between hind gut acidity, the immune system, parakeratosis (thefirst stage of ulcer formation) and physical and physiological changesin the hind gut form the basis of acidic gut syndrome.

Based on these results, there is strong evidence to suggest that acidicgut syndrome is associated numerous other secondary changes developingfrom this condition, such as:

Arthritis and joint conditions where the immune system is implicated;

Susceptibility of the gut to parasitic infections through changes in theepithelium disrupting its integrity and facilitating establishment ofbacteria, fungi and helminths in the gut wall;

Lung infections. Pneumonia is well established as a concurrent diseasewith gastric ulcers in pigs (Kavanagh, 1994) and acid gut syndrome couldbe a predisposing condition for both disease conditions;

Bleeding in the lungs following strenuous exercise results from damageto the alveolar tissue which could be potentiated by changes to theimmune system caused by acidic gut syndrome;

Asthma and skin conditions associated with changes in the immune systemcould be caused or exacerbated by acidic gut syndrome;

Auto-immune conditions could also initiated or exacerbated by acidic gutsyndrome;

Any conditions related to inflammation of the gut such as appendicitis,Crohn's disease, inflammatory bowel disease, colitis and chronicdiarrhoea. Direct effects of acidity and endotoxins on the gut wall maylead to these inflammatory conditions. It is also possible that indirecteffects may predispose development of appendicitis and otherinflammatory conditions in the same way as parakeratosis (stomachulcers) were observed to develop in the current study; and

Reduced feed intake in response to acid accumulation in the gut, shownin this study, could have serious implications for production feedingsystems and prevention of acidic gut syndrome is likely to producesignificant improvements in productivity.

Example 7 Acidic gut syndrome: evidence of an inflammatory responsereleasing the cytokine TNF-α

Background and Hypothesis

It is known that endotoxins are present in the rumen of ruminant animals(Dougherty et al., 1975) and in the hind gut of horses. Experiments inwhich endotoxins have been administered to the gut have not produced anyadverse effects on animal health and it has been concluded that they donot cross the gut wall. For this reason the consequences of fermentativeacidosis in the gut have been explained in terms of the absorption ofacids and systemic acidosis (Dunlop, 1972; Blood et al., 1983, Nocek,1997).

Hypothesis and Implications

The hypothesis on which the current study was based is that theaccumulation of acid and low pH can combine with bacteria and endotoxinsin the gut to produce an inflammatory response which transforms the gutand its contents to a status equivalent to septicaemia in which intactbacteria or their toxins gain access to the host's tissues and thesystemic circulation. TNF-α is known to play a pivotal role ininitiating systemic toxaemic reactions during endotoxaemic sepsis, butits role in mediating effects of acid accumulation in the hind gut hasnot previously been identified. If TNF-α is produced in response tofermentative acid accumulation in the gut, then there is a clear casefor implicating acidic gut syndrome as a contributor to the manyconsequences of systemic cytokine activity known to be associated withelevated levels of TNF-α.

TNF-α

Tumour Necrosis Factor (TNF) is a 17kD polypeptide, which is a member ofthe cytokine family. TNF is also known as cachectin, and referred mostcommonly to by its circulatory form, TNF-α. TNF-α receptors can be foundin almost all cells in the body (Beutler et al., 1985). In smallamounts, TNF-α causes tissue remodelling and local inflammation (Traceyet al., 1989). If large amounts are present it can be released into theblood and act as an endocrine hormone (Abbas et al., 1996), leading tocachexia (wasting), tissue injury and irreversible shock and death(Tracey et al., 1989) (Tracey and Cerami, 1993). TNF-α is also known tobe found in very high levels in patients with inflammatory Bowel Disease(IBD), especially Crohn's disease (Murch et al., 1991; van Deventer,1997) and is suspected to mediate many of the effects of IBD includingcachexia.

When TNF-α is released in large amounts and it acts in the blood,significant levels are also released back into the lumen of the gut.Recently, TNF-α has been reported in the faeces of children with IBD asa marker of intestinal inflammation and disease severity (Braegger etal., 1992); (Nicholls et al., 1993) indicating that mucosally producedTNF-α is also released into the gut. (Braegger et al., 1992) report thatchildren can normally have between 15 and 100 pg TNF-α/g faeces. Inchildren with active Crohn's disease, TNF-α can be as high as 10,000pg/g faeces. In this study, faecal TNF-α was measured in animals fedgrain to determine if a build up of acid in the gut produced TNF-α in asimilar way as occurs in colitis and sepsis.

Experimental Design And Feeding Regime

Animals Housing and Feeding

Six full mouth cannulated cross-bred sheep, weighing approximately 45 kgwere used in the trial. These sheep were housed in individual pens on awood grating floor in an enclosed animal house. All sheep were fed onceper day, and water was available at all times ad libitum. All sheep werefed a basal diet consisting of oaten chaff plus 1% urea and 1% mineralpre-mix (Pfizer Animal Health Pty. Ltd.). At time 0 in the experiment,all sheep were fed a pellet containing high levels of barley with orwithout virginiamycin (VM). The sheep were autopsied 48 hours aftergrain feeding.

Dietary Treatments

All sheep were offered 1000 g of a pellet containing 76% barley, 17%lucerne, 3.5% cottonseed meal, 1% urea and 1.5% limestone at the startof the experiment. The sheep were then offered another 1000 g of thepellet 12 hours later. All sheep consumed all of the pelleted feedoffered. Half of the sheep received the pellets with no dietaryadditives, and the other half received the pellets with virginiamycin(VM) added at 30 mg/kg. Eskalin (Pfizer Animal Health Pty. Ltd., 20 gvirginiamycin/kg) was included at 1.5 g/kg pellets to provide 30 mgVM/kg in the mixed diet. The animals were monitored closely afterreceiving grain and autopsied 48 hours after initial grain feeding.

Samples and Collections

All sheep were fitted with faecal collection bags, which consisted of aPVC ring attached to the posterior of the sheep to which a plastic bagwas attached in order to collect total faecal output. Rumen fluid wastaken via the rumen fistula. Rumen and faecal samples were taken at 4hourly intervals for the first 12 hours after grain feeding. Sampleswere then taken at 24, 36 and 48 hours after initial feeding.

Sample Analysis

Rumen Fluid

Rumen fluid pH was analysed immediately using a portable pH meter(‘Piccolo 2’ ATC pH meter by Hanna Instruments). A sample was also addedto a 20 mL McCartney bottle containing approximately 7 μL ofconcentrated H₂SO₄, and frozen for later analysis of lactic and volatilefatty acids.

Faeces

A representative 20 g sample of faeces was taken into a small vial, anequal volume of water added and mixed well. The faecal pH was thenmeasured immediately on this sample as for rumen fluid. A further samplewas placed in a sterile container and an equal weight of deionised wateradded to it. This was mixed well using a sterile spatula, and thencentrifuged at 15000 rpm for 20 mins. The supernatant was thentransferred using a sterile pipette to 3 individual microfuge tubes andfrozen for later analysis for TNF-α. TNF-α was measured in the faecesusing an ELISA method.

Results

All sheep developed acidosis both in the rumen and in the hind-gut,indicating that the acidosis preventing capacity of VM was exceeded.Rumen pH dropped below 5.0 in 4 of the 6 sheep, while faecal pH droppedto below 5.0 in 3 of the 6 sheep. There were no differences in rumen orfaecal pH between treatment groups (Table 3). When the sheep wereautopsied after 48 hours, the pH along the hind-gut was measured, toestimate a change in pH along the tract towards the faeces from thecaecum.

TABLE 3 Average rumen and faecal pH and faecal lactate (36 and 48 hoursamples) in sheep overfed grain with or without VM (30 mg/kg). P-valuesTreat- Treatment × Measurement Control VM SED ment Time Time Rumen pH5.90 5.84 0.656 ns 0.0001 ns Faecal pH 7.12 7.28 0.768 ns 0.0001 nsFaecal 49.6 28.2 ns 0.02 ns L-lactate Faecal 13.6 7.43 ns 0.05 nsD-lactate

The rumen VFA and lactic acid concentrations are shown in Table 4. TheVM had a significant effect on the pattern of VFA fermentation. Theratio of Propionate to Acetate+(2×Butyrate) was significantly higher insheep fed VM. No treatment effects were observed with respect to lacticacid concentration.

TABLE 4 Summary of rumen volatile fatty acid VFA and lactic acidconcentrations (mmol/L) proportions (mmol/100 mmol) in sheep fed highgrain pellets without any dietary additives, or containing VM (30mg/kg). P-values Treat- Treat- ment × Measurement Control VM SED mentTime Time Total VFA 82.29 81.80 23.575 ns 0.01 ns Ac (%) 62.68 50.9419.930 0.035 0.0001 ns Pr (%) 20.46 34.36 6.365 0.005 0.001 0.006 But (%)11.56 8.75 7.392 ns 0.08 ns L-Lactic acid 5.11 7.33 ns 0.02 ns D-Lacticacid 18.04 25.12 ns 0.04 ns

Faecal TNF-α concentrations are summarised in Table 5. The concentrationof TNF-α in the faeces, did increase significantly from time 0 until 48hours after grain feeding (P=0.019).

TABLE 5 Faecal TNF-α concentrations in 6 sheep overfed a pellet high inBarley and suffering from acute acidosis. Experimental Hour SheepTreatment 0 24 36 48 1 Control 10000 12000 62000 28000 3 Control 2066730000 37000 40000 5 Control 19333 20000 51000 26000 2 VM  9000 1200098333 230667  4 VM 16000 24000 64000 66000 6 VM 12000 17000 25000 76000Averages Control 16667 20667 50000 31333 se  3355  5207  7234  4372 VM12333 17667 62444 124222  se  2028  3480 21184 53300 Average 14500 1916756222 77778 se  2003  2880 10390 31677

Faecal TNF-α was closely related to faecal pH as measured over thecourse of the experiment. FIG. 7 shows the change in faecal pH and TNF-αover the course of the experiment. Faecal pH decreased significantlyover time (P=0.0001) while faecal TNF-α increased significantly over thesame time period (P=0.019). Faecal pH at time 0, 24 and 36 hours wasinversely proportional to faecal TNF-α over the time periods 24, 36 and48 hours.

FIG. 8 illustrates the change in pH of digesta through thegastrointestinal tract to the faeces. It is clear from FIG. 8 thatvirginiamycin has a consistent effect in reducing acid cumulation(increasing pH) in all parts of the digestive tract, and providesevidence for its use in the prevention and control of acidic gutsyndrome in pigs and sheep.

FIG. 8 shows that changes in faecal pH are likely to give a veryconservative estimate of the severity of acidic gut syndrome. Thisobservation is also supported by FIG. 6. In both the sheep and pig data,it is clear that the pH in the caecum and proximal colon is very muchlower than in the rectum or faeces. It is emphasised that the differencebetween the faecal/rectal pH and the caecal/proximal colon is nearly onepH unit (10 fold increase). Therefore, what might appear as a relativelyminor drop in faecal pH from 7.0 to 6.5 is likely to represent a drop incaecal pH from around 6.8 down to 5.5.

This observation is important for two reasons. Firstly, it means thatsince faecal pH normally stays high, even when there is hind gut acidaccumulation, there has been little evidence of any likely problem.Secondly, when faecal pH falls below 6.5 (which it commonly does—seeFIG. 3) there is good evidence that acidic gut syndrome is a potentialproblem.

A further point made in both FIGS. 6 and 8, is that virginiamycin has aconsistent effect in reducing acid accumulation (increasing pH) in allparts of the digestive tract. This provides evidence for its use in theprevention and control of acid gut syndrome. The effect of virginiamycinon pH appears to be less in the faeces than in other parts of the hindgut and it is therefore likely that a prediction of the effects ofvirginiamycin, assessed by sampling faeces alone will give aconservative estimate of the true benefits associated with reduced acidconcentrations.

Discussion

The major finding in this study was the high levels of TNF-oc measuredin the faeces of sheep with fermentative acidosis in the rumen and hindgut. This is the first time that TNF-α has been studied in animals withfermentative acidosis in the gut and it is the first report of elevatedconcentrations of TNF-α in response to acid accumulation in the gut. Itis a very significant addition to our knowledge of the effects offermentative acidosis since it was previously considered that theadverse effects of fermentative acidosis were mediated by absorption ofacids and systemic acidosis based on the absorbed acid (Blood et al.1983).

As described by (Rowe, 1997) and (Ding et al., 1998), there is goodevidence that lactic acid is not absorbed from the rumen or caecum untilthere is severe damage to the gut wall. The new disease condition acidicgut syndrome is in fact based on the adverse effects initiated via lowpH within the gut causing inflammation of the gut wall and thesubsequent release of cytokines which are capable of initiating andexacerbating a wide range of disease conditions well described throughtheir known effects originating from sepsis and other causes ofinflammation.

It has been known for some time that the main mediator of endotoxaemiafrom bacterial infection is through cytokines such as TNF-α (Abbas etal., 1996); (Tracey et al., 1989); (Tracey et al., 1986). This cytokineis prevalent in many conditions including cancer (Balkwill et al.,1987), Cystic Fibrosis (Briars et al., 1995) and HIV infection(Sharpstone et al., 1996). TNF-α is also found in high levels andthought to be one of the main mediators of the effects of irritablebowel disease (IBD) such as Crohn's disease (van Deventer, 1997).Recently, TNF-α levels in faeces of children with active Crohn's diseasehas been used as a marker of intestinal inflammation and diseaseseverity (Braegger et al., 35 1992); (Nicholls et al., 1993).

TNF-α has also been found to be associated with the degradation of manyorgans in the body in which basement membranes are affected, includingliver abscess (Fukui et al., 1993) and respiratory problems (Tracey etal., 1986).

There are also many secondary effects of TNF-α found in many speciesincluding arthritis (Lewthwaite et al., 1995, May, 1997), dermatitis(Nickoloff and Turka, 1993), mastitis and respiratory disease (Godson etal, 1997) that have never previously been related to acid build up inthe gut, but are likely to be initiated or exacerbated by TNF-αinducedfrom acid accumulation in the gut.

The role TNF-α in cachexia or wasting is well established (Tracey et al.1989; van Deventer, 1997) and it is likely that this is partly due toreduced feed intake. The evidence of TNF-α as a result of acidaccumulation indicates the role of cytokines in reduced feed intakeobserved in Example 6 and offers a new approach to managing thesignificant industry problem of variable and low feed intake.

Example 8 Faecal pH in mice with ulcerative colitis Background

TNF is known to be an important mediator of inflammatory bowel diseasein humans (vanDeventer, 1997). A model for inducing ulcerative colitisfor the study of inflammatory bowel disease involves the addition of a5% Dextran Sulphate Solution (DSS) to the water of the mice, and within5 days, the mice show signs of diarrhoea and rectal bleeding associatedwith ulcerative colitis. Dextran sulphate is considered to be anirritant to the gut wall and no link with acid build up in the gut hasbeen previously suggested. The hypothesis behind this study was thatdextran sulphate may in fact provide substrate for rapid fermentation ofthe hind gut and/or that any irritant effect on the gut wall wouldincrease the rate of flow of digesta, thereby decreasing digestion inthe small intestine and increasing hind gut fermentation and acidaccumulation.

The aim of this study was to follow changes in faecal pH during theonset and development of inflammatory bowel disease to determine thepossible role of hind gut acid accumulation on the development ofulcerative colitis

Materials and Methods

Animals and Housing

25 mice were housed in individual pens over 11 days. They were fed abasal diet common to mice consisting of a commercial mouse pellet. Theyhad water available ad libitum.

On day 1 of the trial 15 mice had sodium dextran sulphate included intheir water at 5%. Mice were observed over the next 10 days forincidence of diarrhoea and rectal bleeding as an indication of the onsetof acute colitis.

Treatments

10 animals were used for the control group, in which they remained onthe basal diet with unlimited H₂O with no additives. 15 mice were usedin the colitis group. From day 1 of the trial these mice were fed thebasal diet, but with H₂O containing 5% DSS. This DSS addition has beenpreviously found to cause ulcerative colitis in mice when fed for aperiod of greater than 5 days. These mice remained on this treatment for10 days.

Measurements and Analysis

Total mouse faecal output was collected daily from all mice, and thesamples stored individually in ELISA reaction plates and frozenimmediately in liquid nitrogen. Samples were stored at −20° C. for lateranalysis.

Symptoms of disease were observed and recorded daily for each mouse.Scores from 0 (no symptom) to 5 (severe symptom) were recorded fordiarrhoea and rectal bleeding. These were added together to give TotalDisease Score for each animal for each day. pH Analysis

Individual mouse faecal samples were thawed and diluted with 100 μL ofdeionised water and mixed until confluent. The pH was then recorded onthat sample with an Activon pH probe with Activon pH meter and read to 2decimal places.

Results

Overall, there was no difference between groups in faecal pH. It isinteresting to look at the pH over different periods of the trialhowever in FIG. 9. The faecal pH in the colitis group declinedsignificantly over the period of days 3 to 7 (P=0.0140), and wassignificantly lower than the control group over this period (P=0.0426).After day 5 of the experiment however, there were significant signs ofcolitis with diarrhoea and rectal bleeding both increasing significantly(p=0.0001) as shown in FIG. 10. After this time, the pH of the faecesrose significantly from day 8 to day 10 (P=0.0041), and wassignificantly higher than the control group (P=0.0265).

There was a strong correlation between average pH and average diseasescore for the 10 days of the trial (r²=0.74) as seen in FIG. 10.

Discussion

The initial decrease in faecal pH could be indicative of acid build upin the hind gut particularly as FIGS. 6 and 8 show that relatively minorreduction in faecal pH can reflect a much larger reduction in the pH ofcaecal and colonic digesta. It is however not clear whether hind gutacidosis is a primary or contributory factor in the subsequentdevelopment of ulcerative colitis. The main aspect of the discoveryreported in this Example is in relation to the pattern of pH change andthe similarities with changes seen in response to hind gut acidaccumulation shown in FIG. 7.

After the initial decrease in pH before day 5 when the signs of diseasewere first noted, the pH in the faeces increased significantly from 6.98on day 6 to 7.58 on day 10 (P=0.0041). The pH in the colitis group wassignificantly higher than the control group over this period (P=0.0265).There was a strong correlation found between disease score and observedfaecal pH (r²=0.3157). It is likely that this rise in faecal pH wasprobably due to increased blood mucus secretions from the caecum, largeintestine and colon in response to damage to the epithelial layers fromdisease conditions.

There is important information in the similarities observed between thisstudy in mice and the results in ruminants (see FIG. 7). The rise infaecal pH rising after acid build up in the gut is likely to result fromincreased blood mucus secretions and an increased pH of the faeces couldbe indicative of damage to the gut. Recognising this apparent anomaly ofan increase in faecal pH during the diarrhoea phase of acidic gutsyndrome is important new aspect in understanding and recognising thecondition of acidic gut syndrome.

When colitis occurs in the mouse, damage to the gut wall leads to a risein the tissue levels of TNF, and many of the disease symptoms noted inthe body as a result of the colitis are mediated through tissue levelsof TNF (vanDeventer, 1997). The similarities between patterns of faecalpH change in the mouse in and in sheep with two models (dextran sulphateand grain) causing inflammation in the hindgut provides further evidenceof the links between acidic gut syndrome inflammation to the gut walland the role of inflammatory cytokines exemplified by TNF.

Example 9

Animals or humans with inefficient digestion and absorption of starchand carbohydrate prior to the ileum, may develop chronic acidic gutsyndrome which leads to the gradual development of arthritis andsporadic problems of dermatitis. Treatment in accordance with thepresent invention would initially involve administration ofvirginiamycin (0.4 mg/kg bodyweight per day) and a change in the dietaryregime over the following 2 months in order to stabilise both thearthritis and dermatitis.

Essentially, the change to the dietary regime is designed to minimisethe amount of fermentable carbohydrate which passes through theforestomach, stomach and small intestine of said human or animal, and isavailable for fermentation in the hind gut (ileum, caecum, colon andrectum) of said human or animal.

Typically, the principles on which such a dietary regime may be designedinclude the following:

1. Exclusion or reduction of grains or starch with characteristics ofbeing resistant to gastric digestion and/or intestinal digestion. Forexample, high amylase maize, sorghum and ordinary maize.

2. Exclusion or reduction of grains or grain products with high levelsof non-starch polysaccharides (particularly soluble non-starchpolysaccharides) such as wheat, rye or barley.

3. Ensure that starch-based foods are well cooked to reduce resistanceof starch, and ensure it is broken down or gelatinised to make it morereadily digestible

4. Reduce consumption of barley, rye, wheat or oat-based fermentationproduce, such as beer, which contain more non-starch polysaccharidesthan similar products produced from rice, maize, sorghum or potatoes.

5. Reduce intake of live yoghurts in combination with carbohydratesources containing resistant starch and/or non-starch polysaccharide.For example, yoghurt with muesli.

6. Reduce intake of processed starches used as thickeners in foodpreparation.

7. Avoid a rapid and major change in source and amount of dietarycarbohydrate.

8. Avoid large meals, eat slowly and masticate food well.

Example 10

Children with recurring problems of asthma often may enjoy a dietconsisting predominantly of wheat-based cereal for breakfast and pastafor other meals, washed down with cordial. Treatment in accordance withthe present invention would initially involve administration ofvirginiamycin to stabilise the condition of acidic gut syndrome. This isaccompanied by monitoring of stool pH prior to virginiamycinadministration; and for a treatment period of 4 weeks. Changes indietary habits are difficult to achieve and the introduction of anenzyme mixture by sprinkling (10 g/kg DM of food) over food to assistdigestion in the stomach and absorption from the small intestine, wouldprovide an alternative treatment.

Example 11

A busy executive under significant stress at work may have a largebreakfast each day of muesli and live Lactobacillus yoghurt. There isoften not time for lunch and a large dinner (normally pasta) is quicklyconsumed on the way to evening activities. The executive finds problemsof repeated viral infection (flu and cold sores). Monitoring of faecalpH, endotoxins and TNF reveals higher than normal acidity and detectablelevels of TNF and endotoxins. These changes may be reversed over thefollowing three weeks through treatment with virginiamycin. This may actas a stimulus for the executive to change dietary and eating habits andvirginiamycin could be withdrawn without recurrence of acidic gutsyndrome. A monitoring program (faecal pH and TNF) would be recommendedunder these conditions.

Industrial Applicability

The present invention makes use of a method for the treatment orprophylaxis of acidic gut syndrome resulting from the accumulation ofacid and production of endotoxin in the gastrointestinal tract of ahuman or an animal, said accumulation resulting from the fermentation ofcarbohydrate in the gastrointestinal tract of said human or animal,wherein said method comprises administering to said human or animal aneffective amount of an active agent capable of preventing or controllingacid and endotoxin accumulation in the gastrointestinal tract.

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What is claimed is:
 1. A method for the treatment or prophylaxis ofacidic gut syndrome resulting from the accumulation of acid andproduction of endotoxin in the gastrointestinal tract of a human or ananimal, said accumulation resulting from the fermentation ofcarbohydrate in the gastrointestinal tract of said human or animal,wherein said method comprises administering to said human or animal aneffective amount of an active agent for immunizing the animal against amicroorganism responsible for the fermentation of carbohydrates in thegastrointestinal tract of said human or animal.
 2. The method of claim1, wherein the microorganism is selected from the group consisting of:Aerococcus, Alloiococcus, Carnobacterium, Enterococcus, Lactobacillis,Lactococcus, Leuconostoc, Pediococcu, Streptococcus, andTetragenacoccus.
 3. The method of claim 1, wherein the microorganism isLactobacillis spp. or Streptococcus bovis.
 4. The method according toclaim 1, wherein said microorganism is Streptococcus bovis (Sb-5). 5.The method according to claim 1, wherein the acidic gut syndrome isassociated with a condition selected from the group consisting ofpredisposition to ulceration of the gastrointestinal tract; ulcerationof the stomach; immune conditions associated with localized inflammationof the gut; crohn's disease, appendicitis and colitis; reduced feedintake responsible for cachexia and low efficiency in production feedingsystems.
 6. The method according to claim 1, wherein the acidic gutsyndrome is associated with a condition selected from the groupconsisting of dermatitis; arthritis; rheumatoid arthritis;osteoarthritis; and respiratory tract disorders.
 7. The method accordingto claim 1, wherein the acidic gut syndrome is associated with acondition selected from the group consisting of predisposition tomicrobial and helminth infections of the gut and infection of themammary gland.
 8. The method according to claim 1, wherein the acidicgut syndrome is associated with a condition selected from the groupconsisting of immune disorders causing predisposition to infection bybacteria, fungi or protozoa, cystic fibrosis and cancer.
 9. The methodaccording to claim 1, wherein the acidic gut syndrome is associated witha condition selected from the group consisting of effects on thepancreas, kidneys, thyroid and other organs of the endocrine system; andhomeostasis disorders.
 10. The method according to claim 1, wherein theacidic gut syndrome is associated with a condition selected from thegroup consisting of immune disorders, Alzheimer's disease, impairedreproductive performance; dental caries; viral infections, exacerbationof heat stress; and impaired hair and wool growth.
 11. The methodaccording to claim 5, wherein the condition is irritable bowel syndrome.12. The method according to claim 6, wherein the condition is asthma orpredisposition to bleeding in lungs following strenuous exercise. 13.The method according to claim 7, wherein the condition is mastitis. 14.The method according to claim 5, wherein the condition is diabetes,osteoporosis, or hypertension.
 15. The method according to claim 10,wherein the condition is multiple sclerosis, amyotrophic lateralsclerosis, chronic fatigue syndrome, myasthenia gravis, or herpes.